CN104606085B - A kind of chlorhexidine acetate nano-emulsion gargle of antibacterial and preparation method thereof - Google Patents
A kind of chlorhexidine acetate nano-emulsion gargle of antibacterial and preparation method thereof Download PDFInfo
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Abstract
The present invention provides a kind of antibacterial chlorhexidine acetate nano-emulsion gargle and preparation method thereof.Nano-emulsion gargle of the present invention, is made of the supplementary material of following parts by weight：0.2~1.5 part of chlorhexidine acetate, 8~30 parts of surfactant, 4~15 parts of cosurfactant, 3~20 parts of oil phase, 0.05~1 part of oral cleansing lotion, 34.45~69.0 parts of distilled water.Nano-emulsion of the present invention liquid energy of gargling significantly improves its solubility, and quality is extremely stable, has particularly by the destruction of cell membrane to oral cavity pathogen for streptococcus mutans of representative etc. and is remarkably reinforced inside and outside antibacterial effect.Product of the present invention is easy to use, of low cost, evident in efficacy, not spirituosity, safe and non-toxic, non-stimulated, without side-effects, does not develop immunity to drugs, is a kind of sterilizing oral, sterilizing clean-keeping articles for use.It can be widely applied to the mouth diseases such as periodontitis, gingivitis, canker sore, plaque, dental hemorrhage, halitosis.
Description
Technical field
The invention belongs to field of medicaments, it is related to a kind of nano-emulsion gargle that can treat mouth disease, more particularly to one
Chlorhexidine acetate nano-emulsion gargle of kind antibacterial and preparation method thereof.
Background technology
Since internal environment of oral cavity (humidity, temperature and nutrition condition) is particularly suitable for microbial growth, so oral cavity is
There are most positions for microbe number in human body.Some researches show that：Exist in each gram of tartar (tooth rust) of normal healthy people
10000000000 bacteriums, much larger number of the bacterium than skin surface in oral cavity.Therefore, oral cleaning not in time, can cause halitosis,
The a variety of diseases of various focusing depths represented, canker sore, decayed tooth, periodontosis, so as to seriously endanger people's health.The cleaning in oral cavity becomes people
The first step of health and the important component of daily life.At present, the cleaning usually to oral cavity, though it is most using brush
Tooth, using the external force such as brush, oral rinsing device, oral cleansing lotion between dental floss, toothpick, tooth reach cleaning action, but in oral cavity
The bacterium of growth, particularly peculiar smell are serious, the people of periodontal inflammation, it is impossible to reach and effectively dispel.And brush teeth and be confined to morning
Before sleeping after morning gets up and at night, use is also inconvenient, cleaning not in time, be not thorough, have the drawbacks such as abrasion to tooth, it is difficult to it is full
The oral cleaning requirement of sufficient people.Gargle has progressed into mediocrity's as a kind of new oral cavity cleaning and nursing product
In life, it can not only remove the swill of delay using the impact force that liquid flows in mouth, moreover it is possible to utilize mouthwash
The active ingredient of middle addition removes the pathogenic bacteria in oral cavity.It is packaged in portable Packaging Bottle, can be used at any time,
Can thoroughly overcome oral care not in time, toothpaste is inconvenient to use, cleaning such as is not thorough at the drawback, so as to play cleaning tooth
Tooth, tooth-whitening, remove dental plaque, kill the effect such as oral cavity harmful bacteria, pre- anti-caries, fresh breath.It is especially to such as gum
Certain positive effect is played in the treatment of some mouth diseases such as inflammation, and there is cleaning oral cavity, pre- anti-caries and periodontitis etc. to make
With.Therefore, it is very popular in recent years and widely uses.At present, in the market gargle various types, mainly there is fluorination
The chemistry such as thing, triclosan (the chloro- 2- dihydroxy diphenyl ethers of 2,4,4- tri-), chlorohexidene (chlorhexidine), metronidazole, silver nitrate
The compound preparation of preparation and Chinese herbal medicine.And most widely used fluoride and silver nitrate.At present, though most widely used fluoride
Fluor-apatite protective layer (CaF2) can be formed in enamel surface, strengthen tooth acid-resisting, but it may cause fluorine after
Poisoning, fluorosis of bone, den tal fluorosis, moreover it is possible to cause the generation of a large amount of F resistant Strains, lower curative effect；Silver nitrate can make in dentinal tubule
Nerve fibre cell solidification occurs and forms protective layer, reduce neural reflex, slow down the symptoms such as pain, but can be to soft tissue
It is corrosive and irritation, while Ag+ excessively can be harmful, Ag+ is generated with the reaction of intraoral reducing substances sometimes
Ag causes tooth blackening.And other antibiotic directly suppress or kill oral cavity bacterium, but the abuse of antibiotic easily causes extensively
General bacterial resistance, so as to lessen the curative effect.There is under climbing groundsel, smilax, leaf common compound Chinese medicinal preparation in existing gargle
Pearl, Chinese wingnut leaf, hop etc., but since Chinese herbal medicine extract made from traditional water extraction method, solvent extraction method is inevitable
Can the problems such as low, content is difficult to determine, solvent composition is difficult to remove, gained component is excessively complicated there are active matter content, very
Easily influence it and use mouthfeel.If adding more essence to improve the mouthfeel of product, excessive again may using essence
There is potential threat to the health of human body.
Therefore, though having many various mouthwashes at present, it is all not satisfactory to chase after its final curative effect, at most simply plays temporarily
Slow pain or the effect for cleaning tooth.Many dental patients are difficult to edible cold and hot sour-sweet hard grade food, particularly carious tooth sufferer
Person, it is usually in great pain, it is serious tooth to be pulled out, there are some patients, because there is heredity and pathological factor, and obtaining not
To effective treatment and control, tooth is caused premature loosening occur or come off.For medical point, currently without preferable fast
Prompt treatment amplification and medicine, the department of stomatology is generally more to be gargled using sodium-chloride water solution, interim to solve the problems, such as pain, but up to not
To the purpose of healing, from health care angle for, sold various mouthwashes and medicated toothpaste currently on the market, only served on the time
General health-care effect, it is little to general mouth disease curative effect.And the mouthwash of in the market mostly contains alcoholic content, some are gargled
Alcohol content in saliva is up to 26%, and professor Luo Binximo of the restorative dentistry of University of Newcastle represents, alcohol
The private film for keeping oral cavity moistening itself can be destroyed, makes xerostomia, and then causes halitosis, oral cavity is produced uncomfortable acupuncture
Sense.
Research shows both at home and abroad, and a kind of broad-spectrum antiseptic non-antibiotic class, be now widely used in various apparatuses and operation
Disinfecting drug chlorhexidine acetate to mouth disease to having therapeutic effect.Now research show, its be by with the phosphoric acid in lipopolysaccharides
Carboxyl in salt and chlorhexidine acetate combines, so that interference cell film is transported, small-molecule substance is overflowed extracellular mechanism, so as to lead
Cause bacterial death.But chlorhexidine acetate is low effect disinfectants, and solubility is low (0.2g/100g) in water due to it, it is necessary to
Other preparations compounding can just play due effect.At present only on a small quantity after being dissolved by ethanol, and other Chinese medicines, chemical drugs
Thing (most common fluoride and benzalkonium bromide) compatibility improves its antibacterial activity.But compatibility is used in combination, such as fluoride meeting
To bacterium necessarily great drug resistance, a large amount of F resistant Strains are produced, can yet be serious with fluorine poisoning, fluorosis of bone, den tal fluorosis etc.
Side effect；Benzalkonium bromide is a kind of quaternaries cation surface disinfectant, is now widely used in operation, utensil and place etc.
External sterilizing.The smelly fragrance of its smell, taste is extremely bitter, and aqueous solution is in alkaline reaction, and when shaking produces volume foam, it is impossible to be used in oral cavity
Disinfection, and can cause allergic conjunctivitis, hypopsia, contact dermatitis, with causing evil after several minutes of 3% solution bowel lavage
The heart, cold sweat are lethal eventually to die.The serious side effects of death can also caused as vaginadouche.Therefore, prepare and use non-antibiosis
Plain class, single chlorhexidine acetate component, are not added with ethanol, are gargled with regard to that can significantly improve a kind of nano-emulsion of antibacterial of antibacterial synergistic effect
Oral fluid especially seems important and particularly urgent.
The content of the invention
According to the demand and deficiency in above-mentioned field, the present invention provides a kind of nano-emulsion gargle of antibacterial.This nano-emulsion
Gargle, improves drug solubility using nanometer technology, using non-antibiotic class, single chlorhexidine acetate component, and is not added with
Ethanol, nano-emulsion liquid energy of gargling significantly improve antibacterial synergistic effect, are particularly suitable for carious tooth and other suffering from oral disease.
A kind of antimicrobial caries chlorhexidine acetate nano-emulsion gargle, it is characterised in that be made of the raw material of following parts by weight：
0.2~1.5 part of chlorhexidine acetate, 8~30 parts of surfactant, 4~15 parts of cosurfactant, 3~20 parts of oil phase, oral cavity is clear
New 0.05~1 part of agent, 34.45~69.0 parts of distilled water.
The medicine chlorhexidine acetate, the thing concentration range：0.2~1.5 part.
The surfactant is the one or more in polysorbate or polyoxyethylene fatty acid ester；The polysorbate
For the one or more in Tween-80, Tween-85, polysorbas20, the polyoxyethylene fatty acid ester is polyethylene glycol hydrogenated castor-oil plant
Oil 40, one kind in Emulsifier EL-60 40 or its combination；The effective dose of the component is respectively Tween-80：10~30 parts,
Tween-85：10~30 parts, polysorbas20：8~20 parts, Crodaret 40 (RH40)：10~30 parts, polyoxyethylene
Castor oil 40 (EL40)：10~30 parts.
The cosurfactant is：The monohydric alcohol or polyalcohol of sorbitan fatty acid ester or/and C2~C4；It is described
Sorbitan fatty acid ester is sorbester p37, one kind in sorbester p17 and more than one combinations；The monohydric alcohol of the C2~C4 or
Polyalcohol is any of n-butanol, glycerine, 1,3-BDO, 1,3-PD；The effective dose of the cosurfactant
Respectively Arlacel-85:4~15 parts, sorbester p17：5~15 parts；Glycerine：5~10 parts；Propane diols：4.8~15 parts；1,3- fourth two
Alcohol：5~10 parts, n-butanol：5~10 parts.
The oil phase is any of IPM, GTCC, atoleine, rape oil and soya-bean oil, wherein the oil phase is effective
Amount is respectively IPM：3~20 parts, atoleine：5~15 parts, GTCC：3~15 parts, edible vegetable oil：4~15% parts, edible beans
Oil：5~15%.
The oral cleansing lotion is：Any or its combination in natural peppermint, natural borneol, peppermint oil, menthol；
Its effective dose is respectively natural peppermint：0.05~1 part, natural borneol：0.05~1 part, peppermint oil：0.05~1 part, menthol：
0.05~1 part.
Its particle diameter is 1~100nm after the nano-emulsion gargle water infinite dilution.
The preparation method of above-mentioned nano-emulsion gargle, it is as follows to specifically include step：
(1) each material in composition of raw materials is weighed by proportional quantity；
(2) surfactant, cosurfactant, chlorhexidine acetate stirring are fully dissolved, obtains the solution 1 of clarification；
(3) oil phase of proportional quantity is added into the solution 1 of clarification again, the water of 70% proportional quantity is slowly added under stirring
Phase, the solution 2 clarified；
(4) oral cleansing lotion of proportional quantity is dissolved in and is added after solvent in the solution 2 of clarification.
(5) solution 2 of clarification is sufficiently stirred, until shape in 20 DEG C~25 DEG C water phases for being added slowly to remaining proportional quantity
The nano-emulsion gargle small into clear, viscosity；
(6) and then filtration sterilization, chlorhexidine acetate gargle that is filling, being sealed to different size.
When the oral cleansing lotion is natural peppermint or natural borneol, the solvent is water；When the oral cleansing lotion
For peppermint oil or menthol when, the solvent is propane diols.
Surfactant is that nano-emulsion forms necessary material, its main function is to reduce interfacial tension to form interface
Film, promotes nano-emulsion to be formed；Increase the dissolubility of main ingredient, it is ensured that the stability of preparation.Surfactant is divided into non-ionic table
Face activating agent, cationic surfactant, anion surfactant, block copolymer, zwitterionic surfactant, fluorination
Six major class such as surfactant.Since nonionic surface active agent has nontoxic, good biocompatibility, compare in the solution
Stablize, be not easy to be influenced by strong electrolyte, inorganic salts, soda acid, and, haemocylolysis good with the compatibility of other surfaces activating agent
The advantages of smaller etc. numerous and be subject to apply.Meanwhile in the forming process of nano-emulsion, when the hydrophilic and oleophilic of surfactant
When balancing (HLB) value and equal or close oily required HLB value, the highly stable nano-emulsion of easy forming properties.HLB value is 4
~7 surfactant can prepare w/o type nano-emulsion, and surfactant of the HLB value in 8~18 can prepare O/W type nano-emulsions.
Therefore, the surface active agent tween class and polyoxyethylene fatty acid ester class of the invention for using high hlb, these surfactants,
Nontoxic non-stimulated, good biocompatibility, there is certain trophism, is the preferable auxiliary material for preparing various nano-emulsions.It is preferred that：Tween
80th, polysorbate85, Emulsifier EL-60 (EL40), Crodaret (RH-40).
The formation of nano-emulsion also requires have of short duration negative surface tension.And cosurfactant can live by adjusting surface
Property agent hydrophilic and oleophilic value (HLB) surfactant is had larger absorption in oil-water interface, assist surfactant to reduce boundary
Face tension force, reduces the repulsive force between surfactant molecule；Increase interface motion, reduce interface bending during nano-emulsion formation
Ability, is inserted into interfacial film, promotes the formation of the film of radius of curvature very little, expands nano-emulsion forming region, is conducive to prepare
The nano-emulsion of stable quality.The present invention is its main cosurfactant with current most common spans (85,80), at the same time
Yet selection have the function that enhancing cosurfactant low chain alcohols solvent (glycerine, 1,3- butanol, n-butanol, the third two
Alcohol), infiltration is kept between the oil phase and interfacial film upper surface active agent molecule that can allow used in nano-emulsion and is contacted, and easily
In forming interfacial film with surfactant, so as to be more advantageous to formation and the stabilization of nano-emulsion.
In nano-emulsion forming process, oil is that matching surface activating agent participates in forming stable nano-emulsion, their molecular weight is small,
The effect of small molecule oil phase is similar with cosurfactant, is easily embedded into surfactant, is collectively forming interfacial film therewith, from
And promote the formation of nano-emulsion.When the HLB of surfactant of the present invention according to needed for oil phase is close with surfactant, institute's shape
Into emulsion stabilization principle, select atoleine, edible vegetable oil, GTCC, edible soybean oil, isopropyl myristate etc., this
The equal edible of a little oil phase materials, nontoxic non-stimulated, good biocompatibility, there is certain trophism, is to prepare various nano-emulsions
Major medical auxiliary material.
In the present invention, oral cleansing lotion is added also directed to property：Natural peppermint, peppermint oil, menthol, natural borneol, make it
Mouthfeel it is more preferable.
The chlorhexidine acetate that the present invention uses is a kind of wide spectrum non-antibiotic class antimicrobial, it can be with saliva glycoprotein knot
Close, reduce facing adhesion protein, interference bacterial plaque is formed；In addition, chlorohexidene can also be combined with bacterial cell exo polysaccharides, make bacterium
It is not easy to be adsorbed onto and obtains on film, achievees the purpose that prevention and reduce periodontosis and carious disease.Chlorhexidine acetate is adsorbed in bacterium
The permeability barrier of endochylema film, changes the permeability of bacterial cell membrane, leaks out cellular content, so as to suppress and kill bacterium.
Research report, it is to some staphylococcuses, streptococcus mutans, streptococcus salivarius, Candida albicans, Escherichia coli, anaerobism propionic acid
Bacterium is in extremely sensitive；Isocratic sensitivity in bloodthirsty streptococcus.At present, due to the chlorhexidine acetate extremely low (0.2g/ of solubility in water
100g), it only on a small quantity after being dissolved by ethanol, and limited after other Chinese medicines, chemicals compatibility is used for oral cavity disease at present
In disease.Gargle of the present invention is easy to spread, economic, practical, safe and efficient non-antibiotic medicament or preparation, can be had
Imitate pre- anti-caries.
Gargle of the present invention especially can be by suppressing the key link of cariogenic formation：Produce acid, slightly solubility exocellular polysaccharide, biology
Film is formed such that it well treats and prevents effect with carious tooth, not spirituosity in gargle of the present invention, will not to oral cavity and
The person brings side effect.
The preparation method of the chlorhexidine acetate nano-emulsion gargle of the present invention, it is as follows to specifically include step：
(1) preparation of surfactant phase weighs surfactant by formula rate, with helping surface-active to compound in proportion,
The HLB value of the system is calculated, is stirred.The HLB value of common emulsifying agent can be found in some chemical industry handbooks, such as
Chemical industry publishing house《Chemical products handbook》.Since hydrophile-lipophile balance (HLB) value of surfactant has additive properties, can use
The quality method of average obtains the HLB value of surfactant.For example, after two kinds of surfactant As, B mixing, its mixed surfactant
Hydrophile-lipophile balance HLBAB values be HLBAB=(WAHLBA+WBHLBB)/(WA+WB)
WA in formula, WB --- the quality of blend surfactants A, B；
HLBA, HLBB --- surfactant A, the HLB value of B.
(2) surfactant of the amount and cosurfactant, chlorhexidine acetate stirring are completely dissolved, are clarified
Solution；
(3) according to the HLB value of surfactant phase, selection oil, adjusts its ratio, makes needed for its emulsification for the preparation of oil phase
HLB value is close with the HLB of surfactant phase.The oil phase of the amount is added, the water phase of recipe quantity 70% is slowly added under stirring,
The solution clarified；
(4) the natural peppermint of the oral cleansing lotion of the amount or natural borneol are dissolved in water, or peppermint oil and menthol it is molten
Added after propane diols in prescription.
(5) it is added slowly to recipe quantity distilled water at 20 DEG C~25 DEG C to be sufficiently stirred, until it is small to form clear, viscosity
Nano-emulsion gargle.
(6) then with high speed centrifugation, transmission and grain size analysis detection it is up-to-standard after, filtration sterilization is filling, be sealed to not
The chlorhexidine acetate gargle of same specification.
A kind of antibacterial chlorhexidine acetate nano-emulsion gargle appearance of the present invention is colourless transparent liquid, is had fine
Stability.Ageing stability refer to nanometer emulsion oral liquid room temperature natural trend condition place 1 year, appearance, content, granularity,
Current potential and pH are with time lengthening without significant change occurs.The chlorhexidine acetate nano-emulsion gargle is persistently transparent, does not find muddy
Turbid or precipitation, then illustrate that ageing stability is good.The gargle is placed in test tube, seals, is centrifuged under the rotating speed of 13000r/min
30min clears, the wild effect such as significantly layering and medicine precipitation, flocculation do not occur, by Particle Size Analyzer and thoroughly
Radio microscopy measures particle diameter in 1~100nm scopes.
MIC experiments show, see Fig. 6.The MIC of chlorhexidine acetate nano-emulsion gargle is 0.4ug/ml, chlorhexidine acetate water
The MIC of solution is 0.8ug/ml, and the MIC of compound hibitane acetate solution is 1.0ug/ml.Chlorhexidine acetate nano-emulsion is to resistance
Shape streptococcus drug effect is 2 times of its aqueous solution, is 2.5 times of Chlorhexidine Acetate In Compound.The present invention obtains relatively good antibacterial effect
Fruit is that the present invention uses suitable prescription and proportioning, adds suitable cleaning agent, is prepared using nanometer technology and technique anti-
Bacterium gargle.These effects may is that the common collaboration using suitable surfactant, cosurfactant, oil phase, freshener
Effect.
Chlorhexidine acetate nano-emulsion gargle is to Escherichia coli, candida albicans, streptococcus mutans, Bacillus acidi lactici
Testing result Fig. 8 of MIC.As a result find out, nano-emulsion gargle of the invention is to Escherichia coli, candida albicans, deformation chain
The MIC of coccus and Bacillus acidi lactici is respectively 0.8ug/ml, 3.2ug/ml, 0.4ug/ml, 3.2ug/ml.Chlorhexidine acetate folk prescription and
The micro emulsion disinfectant of compound is 3.2ug/ml respectively to Escherichia coli, candida albicans, streptococcus mutans and Bacillus acidi lactici
With 1.6ug/ml, 6.4ug/ml and 4.8ug/ml, 1.6ug/ml and 0.8ug/ml, 6.4ug/ml and 3.2ug/ml.The present invention's
Nano-emulsion gargle is acetic acid chlorine respectively to Escherichia coli, candida albicans, streptococcus mutans and Bacillus acidi lactici antibacterial effect
Own order side and 4 times of compound micro emulsion and 2 times, 2 times and 1.5 times, 4 times and 2 times, 2 times and 1 times.Patent of the present invention is in large intestine bar
Bacterium, candida albicans, streptococcus mutans, the antibacterial activity of Bacillus acidi lactici are micro- apparently higher than chlorhexidine acetate folk prescription and compound
Breast.Patent of the present invention obtain preferable antibacterial effect be the present invention using suitable prescription and proportioning (only in prescription, acetic acid
Chlorhexidine folk prescription and compound micro emulsion have selected that toxicity is big, volatile ethyl acetate, and the present invention have selected without volatility,
The oil phase of safer edible (medicinal)), suitable cleaning agent is added, antibacterial is prepared using nanometer technology and technique and is gargled
Oral fluid.These effects may is that to be acted synergistically using suitable surfactant, cosurfactant, oil phase, the common of freshener
To improve the antibacterial effect of gargle.
It is of the invention compared with other prior arts, there is following beneficial effect：
1. solving chlorhexidine acetate indissoluble sex chromosome mosaicism, prepare chlorhexidine acetate nano-emulsion gargle acetic acid can be greatly improved and wash
Must safe solubility, there is good solubilization.
2. the chlorhexidine acetate nano-emulsion gargle prepared has, viscosity is low, stability is good, it is dispersed it is strong, absorb it is rapid etc.
Feature, medicine are wrapped in kernel oil phase, also have sustained release and targeting, and can improve the bioavilability of medicine, extend
Medicine half-life period in vivo, heighten the effect of a treatment, reduce toxic side effect.
Between 1~100nm, 3. average grain diameter is the diameter of aspirin particle of the chlorhexidine acetate nano-emulsion gargle of the present invention
63.13nm, is that chlorhexidine acetate is dissolved in surfactant and cosurfactant, is titrated to distilled water uniform, transparent
Nano-emulsion.
4. it is formula and method simple possible that the present invention uses, of low cost, can be in order to large-scale industrial production.This
Invention product is easy to use, evident in efficacy, not spirituosity, and side effect will not be brought to oral cavity and the person, and palatability is good.
5. it can thoroughly penetrate Dental Plaque nucleus to the present invention, active ingredient is directly acted on into its kernel, killing is drawn
The anaerobic bacterias of various mouth diseases, Candida albicans, staphylococcus glucose coccus, pyococcus, streptococcus mutans etc. is played to cause
Germ simultaneously controls its growth, reduces hospital-acquired infection and oral cavity postoperative infection, sterilization shield tooth, eliminates oral peculiar smell.Pass through sterilization
Kinetic results show that it acts on streptococcus mutans after five minutes, the bacterium with regard to that can kill 95%；
6. the present invention can quickly kill cause periodontitis, gingivitis, canker sore, plaque, dental hemorrhage, halitosis cause a disease
Bacterium, it is safe and non-toxic, non-stimulated, it is without side-effects, do not develop immunity to drugs, be sterilizing oral, sterilizing clean-keeping articles for use, it may also be used for artificial tooth
Immersion and sterilizing.
Brief description of the drawings：
Fig. 1 is chlorhexidine acetate nano-emulsion gargle prescription screening figure；
Fig. 2 is the drafting of chlorhexidine acetate nano-emulsion gargle pseudo-ternary phase diagram；
Fig. 3 is chlorhexidine acetate nano-emulsion gargle qualification result figure；
Fig. 4 is chlorhexidine acetate nano-emulsion gargle HPLC content detection figures；
Fig. 5 is chlorhexidine acetate nano-emulsion gargle stability test result；
Fig. 6 is chlorhexidine acetate nano-emulsion gargle to streptococcus mutans MIC result figures；
Fig. 7 for chlorhexidine acetate nano-emulsion gargle it is antibacterial to streptococcus mutans and sterilization kinetic results figure；
Fig. 8 detects comparing result figure for chlorhexidine acetate nano-emulsion gargle to different bacterium MIC；
Fig. 9 produces streptococcus mutans acid and glucan for chlorhexidine acetate nano-emulsion gargle influences result figure；
Figure 10 is influence of the chlorhexidine acetate nano-emulsion gargle to streptococcus mutans biofilm formation；
Figure 11 sees streptococcus mutans biofilm surface form atomic force microscope for chlorhexidine acetate nano-emulsion gargle
Examine result figure；
Figure 12 is internal pharmacodynamic evaluation of the chlorhexidine acetate nano-emulsion gargle to streptococcus mutans；
Figure 13 is that chlorhexidine acetate nano-emulsion gargle evaluates the cell membrane disruption of streptococcus mutans.
Embodiment
To better illustrate the object, technical solutions and advantages of the present invention, by the description of embodiment to this hair
It is bright to be described further, but it is limitation of the present invention that this, which is not, and those skilled in the art's basic thought according to the present invention can
To make various modifications or improvement, but without departing from the basic thought of the present invention, within the scope of the present invention.
Embodiment：
Embodiment 1：Chlorhexidine acetate nano-emulsion gargle (100g)
1) chlorhexidine acetate 0.5g, propane diols 4.8g, Tween-80 19.6g, IPM 6g, natural peppermint 0.1g, steaming are weighed
Distilled water 69g.
2) surfactant, cosurfactant and chlorhexidine acetate of the amount are mixed, and rotating speed for 50~
100r/m constant temperature blender with magnetic force stirs, and allows chlorhexidine acetate to be completely dissolved；
3) 25 DEG C of waters bath with thermostatic control of oil phase of the amount are added, and are 100~200r/m constant temperature blender with magnetic force in rotating speed
Stirring, is slowly added to about 70% recipe quantity distilled water, the solution clarified；
4) the natural peppermint of the oral cleansing lotion of the amount is dissolved in water, or peppermint oil and menthol be dissolved in after propane diols plus
Enter in prescription.
5) it is added slowly to recipe quantity distilled water at 20 DEG C~25 DEG C to be sufficiently stirred, until it is small to form clear, viscosity
Nano-emulsion gargle.
6) then with high speed centrifugation, transmission and grain size analysis detection it is up-to-standard after, filtration sterilization is filling, be sealed to not
The chlorhexidine acetate gargle of same specification.
The nano-emulsion mouthwash formulation appearance colorless of the chlorhexidine acetate of preparation, clear, particle diameter between 1~100nm,
It is not stratified after 13000rpm, 10min high speed centrifugation, drugloading rate 0.5%, envelop rate 90%, after room temperature is placed 1 year, nanometer
Breast is separated out without obvious flocculation, layering and medicine.
Embodiment 2
Weigh chlorhexidine acetate 0.5g, propane diols 15g, Tween-80 30g, medicinal IPM 20g, natural peppermint 0.05g, steaming
Distilled water 34.45g.
Embodiment 3
Weigh chlorhexidine acetate 0.5g, glycerine 5g, Tween-85 30g, Arlacel-80 4g, medicinal IPM 10g, natural borneol
0.05g, distilled water 50.45g.Preparation method is the same as embodiment 1.
Embodiment 4
Weigh chlorhexidine acetate 1.0g, glycerine 5g, Tween-80 20g, Arlacel-80 10g, medicinal GTCC 15g, natural thin
Lotus 1.0g, distilled water 48g.Preparation method is the same as embodiment 1.
Embodiment 5
Weigh chlorhexidine acetate 1.0g, glycerine 10g, Tween-85 8g, Tween 80 20g, medicinal IPM 3g, menthol
1.0g, distilled water 57g.Preparation method is the same as embodiment 1.
Embodiment 6
Weigh chlorhexidine acetate 1.5g, propane diols 5g, Tween-80 10g, Tween-20 20g, medicinal liquid paraffin 15g,
Peppermint oil 1.0g, distilled water 47.5g.Preparation method is the same as embodiment 1.
Embodiment 7
Weigh chlorhexidine acetate 1.0g, n-butanol 5g, RH-40 30g, Arlacel-85 4g, medicinal paraffin 10g, peppermint oil
0.05g, distilled water 49.95g.Preparation method is the same as embodiment 1.
Embodiment 8
Weigh chlorhexidine acetate 1.0g, 1,3 butanediol 15g, Tween-85 20g, medicinal IPM 10g, menthol 0.05g,
Distilled water 53.95g.Preparation method is the same as embodiment 1.
Embodiment 9
Weigh chlorhexidine acetate 0.2g, propane diols 10g, Tween 80 20g, Tween-85 10g, medicinal IPM 10g, natural ice
Piece 1g, distilled water 48.8g.Preparation method is the same as embodiment 1.
Embodiment 10
Weigh chlorhexidine acetate 0.2g, propane diols 15g, EL-40 30g, medicinal IPM 15g, distilled water 39.8g.Preparation side
Method is the same as embodiment 1.
Embodiment 11
Weigh chlorhexidine acetate 1g, EL-40 10g, RH-40 10g, Arlacel-85 10g, medicinal liquid paraffin 5g, peppermint
Oily 0.5g, distilled water 63.5g.Preparation method is the same as embodiment 1.
Embodiment 12
Weigh chlorhexidine acetate 0.5g, EL-40 20g, medicinal GTCC 10g, peppermint oil 1.0g, distilled water 68.5g.Prepare
Method is the same as embodiment 1.
Embodiment 13
Weigh chlorhexidine acetate 1.0g, glycerine 5g, EL-40 30g, Arlacel-85 15g, soya-bean oil 10g, natural borneol 1.0g,
Distilled water 38g.Preparation method is the same as embodiment 1.
Embodiment 14
Weigh chlorhexidine acetate 1.0g, glycerine 5g, RH-40 30g, Arlacel-80 15g, rape oil 10g, peppermint oil 0.1g, steaming
Distilled water 38.9g.Preparation method is the same as embodiment 1.
Embodiment 15
Weigh chlorhexidine acetate 1.0g, RH-40 15g, EL40 15g, Arlacel-80 15g, rape oil 4g, peppermint oil 1g, steaming
Distilled water 49g.Preparation method is the same as embodiment 1.
Embodiment 16
Weigh chlorhexidine acetate 1.0g, RH-40 15g, Tween 80 15g, the 15g of glycerine -80, rape oil 15g, natural peppermint
1.0g, distilled water 38g.Preparation method is the same as embodiment 1.
Embodiment 17
Weigh chlorhexidine acetate 1.0g, polysorbate85 20g, Tween 80 15g, -80 15g of 1,3 butanediol, GTCC 4g, thin
Lotus brain -0.5g, distilled water 44.5g.Preparation method is the same as embodiment 1.
Embodiment 18
Weigh chlorhexidine acetate 1.0g, EL-40 10g, RH-40 10g, 1,3 butanediol 5g, soya-bean oil 15g, natural peppermint
1.0g, distilled water 57g.Preparation method is the same as embodiment 1.
Embodiment 19
Weigh chlorhexidine acetate 1.0g, polysorbas20 8g, Tween-85 25g, n-butanol 5g, GTCC 4g, natural borneol
1.0g, distilled water 56g.Preparation method is the same as embodiment 1.
Embodiment 20
Weigh chlorhexidine acetate 1.0g, Tween-20 15g, Tween-80 15g, n-butanol 10g, soya-bean oil 3g, natural borneol
0.5g, distilled water 55.5g.Preparation method is the same as embodiment 1.
Experimental example
Test material：Chlorhexidine acetate (content 98.3%, the safe Pharmaceuticals Ltd in Jinzhou, Liaoning nine)；Chlorhexidine acetate pair
According to product (Nat'l Pharmaceutical ＆ Biological Products Control Institute)；Crodaret (RH40), Emulsifier EL-60 (EL40), moral
BASF AG of state produces, and purchases to Beijing Feng Lijingqiu commerce and trade Co., Ltd；CRODA companies of GTCC Britain produce, and purchase to north
Capital Feng Lijingqiu commerce and trade Co., Ltd；Soya-bean oil, peanut oil are purchased to Shandong Lu Hua Co., Ltds；Tween 80, polysorbate85, department
Disk 80, sorbester p37, polysorbas20, glycerine, propane diols, atoleine, 1,3 butanediols, n-butanol are purchased to Chinese Shanghai Chinese medicines group.
Brain-heart infusion medium (Brain heart infusion, BHI) (BD, New Jersey, USA)；24 porocyte culture plates
(Costar 3524,USA)；Coverslips(Thermo,Rochester,USA).Strain：Streptococcus mutans UA159 (ATCC
700610) (ATCC, Virginia, USA), Escherichia coli (ATCC 8099), candida albicans (ATCC 10231) equal ATCC
Purchase, laboratory, which preserves, to be provided；Bacillus acidi lactici is separated from southwestern hospital clinical, and laboratory, which preserves, to be provided.SD rats, SPF are female
Property, 3 week old, are purchased to Chongqing Institute of Chinese Medicine.
Instrument and equipment：Transmission electron microscope (Dutch PHILIPS companies)；Current potential and Particle Size Analyzer (NanoZS90, Britain Ma Er
Wen companies)；HPLC (Waters-E2695, the U.S.)；ND-1000 ultraviolet specrophotometers (NanoDrop companies of the U.S.)；Enzyme mark
Instrument (Biorad 6.0, Japan), micro- aerobic incubator (Thermo Scitinfic), bacterial colony counting instrument (Shineso
Science＆Technology Co., Ltd., Zhejiang Hangzhou)；Scanning electron microscope (AMRAY1000B, USA)；Atomic Mechanics microscope
(the existing instrument of the National Immunization preparation technique Engineering Research Center such as (IPC-208B, University Of Chongqing).
1. the screening of the optimal prescription of nano-emulsion gargle of chlorhexidine acetate
(1) screening of surfactant and cosurfactant：
With method Surfactant Tween 80, polysorbate85, polysorbas20, the polyoxy of screening chlorhexidine acetate nano-emulsion oil phase
Ethylene castor oil (EL40), Crodaret (RH40) and cosurfactant (CoSF), i.e. sorbester p17, sapn
85 and ethanol, glycerine, propane diols same method screening chlorhexidine acetate saturated concentration.
To above-mentioned surfactant, surfactant, oil phase the selection result, see Fig. 1 a to Fig. 1 c.Live to above-mentioned surface
The solubility test of property agent the result is that：Solubility size is Tween 80 (16.16 ± 0.82mg/g), polyethylene glycol hydrogenated castor successively
Sesame oil (RH40) (13.23 ± 0.56mg/g), Emulsifier EL-60 (EL40) (10.28 ± 0.17mg/g), polysorbate85 (8.45
± 0.08mg/g), polysorbas20 (4.23 ± 0.06mg/g), Fig. 1 a.The solubility test result of above-mentioned cosurfactant is from big
To it is small as：Ethanol (9.60 ± 0.31mg/g), propane diols (7.73 ± 0.30mg/g), sorbester p37 (4.35 ± 0.11mg/
G), sorbester p17 (3.95 ± 0.03mg/g), n-butanol (2.56 ± 0.09mg/g), 1,3 butanediols (2.09 ± 0.46mg/g), are shown in
Fig. 1 b.
(2) screening of oil phase：
Select 5 kinds of different carbon chain length such as IPM (C14), GTCC (C18), atoleine (C16~C20), soya-bean oil, rapeseed oil
Short oil, 20 DEG C of viscosity sizes are followed successively by atoleine (110~230mPas), GTCC (25.0~33.0mPas), soya-bean oil
(8.5mPas), IPM (5~6mPas), rape oil (8.5mPas).Take above-mentioned 5 kinds of oil appropriate respectively, put respectively in conical flask with cover,
Excessive chlorhexidine acetate is added, 25 DEG C of water-baths, shake 24h and reach balance.Chlorhexidine acetate is measured in various oil with HPLC methods
Solubility in phase.
Fig. 1 c the result shows that：IPM (C14), atoleine, GTCC, soya-bean oil, rapeseed oil be respectively 3.08 ± 0.15mg/g,
2.27 ± 0.09mg/g, 1.53 ± 0.08mg/g, 0.89 ± 0.06mg/g, 0.52 ± 0.05mg/g, IPM solubility most
Greatly, it is 3.08mg/g.This may be related with viscosity with the carbochain length of IPM, is conducive to distribution and dissolving of the medicine in oil phase.
Therefore, optimal oil phases of the IPM as chlorhexidine acetate nano-emulsion is selected.All oil phase, surfactant and surface is helped to live
Property agent has larger solubility (being more than 0.2mg/ml), and nano-emulsion requirement can be prepared by meeting.But since ethanol has volatilization
Property and have strong impulse to oral mucosa, selection except he with for all oil phases, surfactant and help surface-active
Agent, but according to its maximum pseudo-ternary phase diagram region, finally determine that its surfactant and cosurfactant interworking are more excellent
It is combined as：Tween 80/sorbester p17, polysorbate85/sorbester p37, RH40/ propane diols, EL40/ propane diols.
(3) drafting of pseudo-ternary phase diagram
With filtering out surfactant and cosurfactant, Comprehensive Designing experimental group, by 9:1、8:2、7:3、6:4、5:
5、4:6、3:7、2:8、1:9 (mass ratioes of SF and CoSF), weigh SF-CoSF, add selected oil phase, and be vortexed concussion, drop
Add water, observe the change of each group, to centrifuge (13,000rpm, 30min) stability and particle diameter as main evaluation criterion, so that
Determine to form the SF and CoSF of nano-emulsion.With surfactants/cosurfactants (mixed surfactant), oil phase and go
3 vertex of the ionized water as equilateral triangle, draw pseudo-ternary phase diagram, and the point in phasor each edge represents corresponding two groups respectively
/ proportionate relationship, phasor any point represents the mass percentage of each component in corresponding system.According to oil, water, mix
Mass fraction of the surfactant in critical point is closed, draws pseudo-ternary phase diagram, determines maximum nano-emulsion area.The evaluation of nano-emulsion
Standard evaluation criterion is：The liquid of preparation is in clear or translucent；There is blue-opalescent；High speed centrifugation (13 000r/m,
After 30min) centrifuging, stablize not stratified；There is Tyndall phenomenon after directional light incidence；Pass through transmission electron microscope and laser particle size
Instrument, detects emulsion droplet between 1~100nm.If the mixing liquid prepared is creamy white, there is scattering phenomenon after directional light incidence,
It is then emulsion；If clear, stiff, for gel.At the same time liquid crystal can be also produced in phase in version process.There is refractive power using liquid crystal
In the presence of in petrographic microscope detection, it may appear that light and shade changes, so often using it to distinguish liquid crystal and gel, is Ru shinny
Liquid crystal, not shinny gel.
Pseudo-ternary phase diagram it is drafting as a result, seeing Fig. 2 a to Fig. 2 d.It can be seen that from Fig. 2 a to Fig. 2 d and work as surfactant
During with ratio (Km) value increase of cosurfactant, the region of nano-emulsion first increases is reduced afterwards.When its ratio be 4:1, it is received
The region of rice milk is maximum, when its ratio be 2：1, its nano-emulsion region is minimum.According to pseudo-ternary phase diagram as a result, so that it is determined that most
Optimal ratio is 4:1.
2. prepared by chlorhexidine acetate nano-emulsion gargle
According to Sequential design thought, Yi Shui, oil, SF/CoSF are three-phase, and pseudo-ternary phase diagram is drawn using titration.Selection
Centrifuge (13,000rpm, 30min) to stablize, diameter is prescription candidate regions in the nano-emulsion area of 1~100nm.Accurately weigh recipe quantity
Chlorhexidine acetate, (surfactant is above-mentioned with cosurfactant for the surfactant of recipe quantity and cosurfactant
Screen 4 kinds more excellent interworkings combinations), the oil phase of recipe quantity, first add about 70% aqueous solution or buffer solution, stirring is equal
It is even, water or buffer solution are finally gradually added, up to the nanoemulsions of clear.It can be seen that from Fig. 3 a, the acetic acid of preparation is washed must
The faint yellow clear liquid of Turner rice milk appearance.The final chlorhexidine acetate for showing to prepare according to pseudo-ternary phase diagram is drawn is received
The optimization formulation of rice milk is：Tween 80：Propane diols：IPM (19.2%:4.8%:6%), chlorhexidine acetate concentration 0.5% and water
56.5%, peppermint oil 5%.Following performance detection such as shows the optimized prescription used.
3. form, granularity and the potentiometric detection of acetic acid nano-emulsion gargle
A small amount of nano-emulsion is appropriate, after being diluted with water 100 times, drops in and is covered with the copper mesh for supporting film, with filter after static 10min
The scraps of paper blot, then 2% phosphotungstic acid (pH 7.4) solution is added dropwise in negative staining 3min on copper mesh, volatilize naturally, use transmission electron microscopy
Sem observation and photographs, the result is shown in Fig. 3 b.Take chlorhexidine acetate nano-emulsion gargle appropriate, used after being diluted with appropriate distilled water
Laser granulometry is measured, and particle diameter and the size distribution of nano-emulsion are shown in Fig. 3 c and Fig. 3 d.
Fig. 3 a the result shows that, rounded under Electronic Speculum, inside is oil phase, sees that Fig. 3 b under Electronic Speculum, are randomly selected not
Less than 500 drops measure particle diameter, obtain nano-emulsion droplet size range in 1~100nm, average grain diameter 63.0nm.Fig. 3 b are tied
Fruit shows, tests the chlorhexidine acetate nano-emulsion average grain diameter 63.13nm of preparation.Particle less than 70nm accounts for 70%；It is less than
The particle of 90nm accounts for 95%, it is seen that the particle size distribution range of obtained nano-emulsion gargle is narrow, and particle diameter is than more uniform.Fig. 3 d results
Show, test the chlorhexidine acetate nano-emulsion average potential -67.13nm of preparation, belong to stabilising system scope.
4. quality testing and the Detection of Stability of chlorhexidine acetate nano-emulsion gargle
Chlorhexidine acetate HPLC detection methods are established：Chromatographic condition：HPLC:Waters(E2695,USA)；Chromatographic column：
ZORABX SB-C18(5um,4.6mm×250mm)：Sample size：10ul；Mobile phase：Acetonitrile：0.02M-K2HPO4 (pH=2.0)
(70/30,v/v)；Detection wavelength：275nm；Column temperature：20℃.Chlorhexidine acetate reference substance is dissolved into 0 with methanol, 25,50,
75、100、200ng/mL.Under above-mentioned chromatographic condition, its peak area of various concentrations is detected, according to various concentrations and peak area,
The calibration curve equation of drafting.Chlorhexidine acetate nano-emulsion gargle 0.2mL, 13 000r/m, 30min are taken, rear precipitation adds
10ml ethanol, 0.22um filtering with microporous membrane HPLC measure its amount do not wrapped up；Supernatant adds the absolute ethyl alcohol of two volumes, shakes
After even demulsification, 13 000r/m, 30min, take supernatant precipitation to be separately added into 10ml ethanol, 0.22um filtering with microporous membrane HPLC
Measure the amount do not wrapped up.Its envelop rate and drugloading rate are calculated with formula.
Drugloading rate=(precipitation capacity after supernatant+demulsification after demulsification) × 100%/sample gross mass.
Envelop rate=(actual drug drugloading rate/theoretical drug drugloading rate) × 100%
Chlorhexidine acetate reference substance, chlorhexidine acetate nano-emulsion gargle HPLC figures appearance time it is basically identical,
Near 4.87min.The standard curve of drafting, is shown in Fig. 4, and linear relationship is good, calibration curve equation Y=14782X-17189
(R2=0.9963, linear equation scope 25-1000ug/mL), there is higher correlation and wider detection range, blank is received
Rice milk does not produce obvious peak at this, illustrates that its separating effect is preferable.Because of the detection method, this can be used for HPLC detection acetic acid
The envelop rate and drugloading rate of chlorohexidene.According to standard curve, the drugloading rate for measuring chlorhexidine acetate nano-emulsion gargle is 5mg/
Ml, its envelop rate are more than 90%.
Take chlorhexidine acetate nano-emulsion gargle 3 batches, 3 parts of every batch of, places 12 under the conditions of 25 DEG C of relative humidity 60 ± 5%
Month.Every 3 months sampling once, respectively at 0,30,60,90,120,180,360 day, first observe high speed centrifugation (13,000rpm,
Cosmetic variation before and after 10min)；And its content is detected with HPLC, current potential and granularity Nano ZS90 current potentials and grain size analysis
Instrument detects, its pH value is surveyed with pH meter.
Stability experiment the result shows that, chlorhexidine acetate nano-emulsion gargle is in above-mentioned institute's having time, in high speed centrifugation
Front and rear appearance all occurs significantly to change, and obvious flocculation, layering and medicine precipitation phenomenon also occurs without generation.It is contained
Amount, granularity and current potential, pH measure that the result is shown in Fig. 5.As can be seen from Figure 5, its content, granularity and current potential, pH are obvious without occurring
Change.The above results fully confirm that the chlorhexidine acetate nano-emulsion quality that it is prepared is highly stable.
Since the pathogenic bacteria in oral cavity are numerous, and streptococcus mutans is the main generally acknowledged pathogenic bacteria in oral cavity, following research
Just studied by representative of streptococcus mutans.
5. antibacterial synergistic effect of the chlorhexidine acetate nano-emulsion to streptococcus mutans
Experiment material and packet：0.2% chlorhexidine acetate aqueous solution group (writes a Chinese character in simplified form CHX, weighs the addition of 0.2g chlorhexidine acetates
It is complete to 100g distilled water, stirring and dissolving), 0.5% chlorhexidine acetate nano-emulsion gargle group (write a Chinese character in simplified form CNE, it is using the present invention
Preparation process, optimized prescription be：Tween 80：Propane diols：IPM (19.2%:4.8%:6%, i.e. specific embodiment 1),
Chlorhexidine acetate concentration 0.5% and water 56.5%, peppermint oil 5%).Blank group (writing a Chinese character in simplified form, MNE), Chlorhexidine Acetate In Compound are gargled
Liquid group (is write a Chinese character in simplified form, FCHX, with reference to 96115216.3 major pharmaceutical component of Chinese patent：0.2% sodium fluoride, 0.02% acetic acid is washed must
Thailand, 0.01% natural sweetener and 0.075% essence, preparation method are the patent of invention Shen according to Application No. 96115261.3
Formula each component and its dosage that please be disclosed, and the dental caries treating agent prepared according to its disclosed method).
The measure of MIC：With reference to preliminary experiment early period, by chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion gargle,
Chlorhexidine Acetate In Compound gargle is diluted to containing chlorhexidine acetate 1,2,4,6,8,10ug/ml respectively.By UA159
The bacterium solution of (Streptococcus mutans ATCC700610) culture is adjusted to OD=1.0.Draw and dilute 1 with BHI:10000
Bacterium solution 1.8ml again, by above-mentioned various concentrations chlorhexidine acetate solution 0.2ml, is put into 37 DEG C of micro- aerobic incubators, cultivates 24h,
200ul liquid is removed per hole, its absorption values is detected at 595nm wavelength with microplate reader.
MIC experiments show, see Fig. 6.The MIC of chlorhexidine acetate nano-emulsion gargle is 0.4ug/ml, chlorhexidine acetate water
The MIC of solution is 0.8ug/ml, and the MIC of compound hibitane acetate solution is 1.0ug/ml.Chlorhexidine acetate nano-emulsion is to resistance
Shape streptococcus drug effect is 2 times of its aqueous solution, is 2.5 times of Chlorhexidine Acetate In Compound.
The measure of MBC：The bacterium solution 5ul of above-mentioned MIC cultures is drawn, instills BHI solid culture plates, L rod coatings, are put into 37 DEG C
Micro- aerobic incubator, cultivates 24h, observes the growing state of bacterium.
MBC the result shows that, the BHI tablets of the chlorhexidine acetate nano-emulsion gargle of 0.4ug/ml do not grow bacterium；
The BHI tablets of the chlorhexidine acetate aqueous solution of 0.8ug/ml do not grow bacterium；The BHI of the compound chlorhexidine gargle in vitro of 1ug/ml
Tablet does not grow bacterium.Therefore, the MBC of chlorhexidine acetate nano-emulsion is 0.4ug/ml, and the MBC of chlorhexidine acetate aqueous solution is
0.8ug/ml, the MBC of compound hibitane acetate solution is 1.0ug/ml.MIC and MBC's as a result, it was confirmed that chlorhexidine acetate nanometer
Breast is remarkably improved to Streptococcus mutans activity.
Sterilize dynamics：The ATCC700610 bacterium solutions cultivated are adjusted to OD=0.6.Draw ATCC700610 bacterium solutions
1.8ml, by 2,4,8ug/ml chlorhexidine acetate aqueous solutions group, chlorhexidine acetate nano-emulsion gargle group, be put into 37 DEG C it is micro- aerobic
Incubator, cultivates 0,5,10,15,30,60,120,240,480 and 720min.After being incubated different time, 0,10 is diluted with BHI1,
102, 103With 104Times, take 5 μ L dilute solutions to be placed on 37 DEG C of 24h to BHI tablets, counted with Automatic Colony Counter.
Sterilize dynamics Fig. 7 a the result shows that, it is observed that nano-emulsion gargle have it is faster and more powerful than CHX
Bactericidal activity.Sterilization kinetic results show that antibacterial effect has notable dose-dependent effect with chlorhexidine acetate.Result of study
Show, the concentration C NE of 0.8 μ g/mL has quick (5min) bactericidal effect to streptococcus mutans than CHX (73.33%)
(95.07% bacterial death).And when the chlorhexidine acetate nano-emulsion gargle of 0.8 μ g/mL can be in 480min by bacterium whole
Kill.After chlorhexidine acetate nano-emulsion gargle effect 5min, time-killing kinetic curve substantially reduces, and chlorhexidine acetate is water-soluble
Liquid is just substantially change in 15min.As a result, it was confirmed that chlorhexidine acetate nano-emulsion gargle is more notable than its aqueous solution to shorten that its is right
Streptococcus mutans act on.
Antibacterial dynamics：Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion gargle, blank group are diluted respectively
Into containing chlorhexidine acetate 2,4,6,8,10ug/ml.The ATCC700610 bacterium solutions cultivated are adjusted to OD=1.Draw
ATCC700610 dilutes 1000 times of bacterium solution 1.8ml with BHI fluid nutrient mediums, by above-mentioned various concentrations chlorhexidine acetate solution
0.2ml, is put into 37 DEG C of micro- aerobic incubators, culture 0,10,20,30,60,120,240,360,480min, 200ul is removed per hole
Liquid, its absorption values is detected with microplate reader at 595nm wavelength.
Antibacterial kinetic results, are shown in Fig. 7 b.From Fig. 7 b, with the extension of time, its OD gradually rises, as culture 60min
When, significant change do not occur for the OD of bacterium, and when chlorhexidine acetate nano-emulsion does not occur significantly to become in 240min with 60min yet
Change, and chlorhexidine acetate aqueous solution in 240min apparently higher than its nano-emulsion.These show that chlorhexidine acetate nano-emulsion is than its water
Solution significantly extends its and antibacterial makees the time.
Above-mentioned MIC, MBC its MIC, MBC, sterilization and antibacterial dynamic test are as a result, it was confirmed that chlorhexidine acetate nano-emulsion
There is significant antibacterial activity to streptococcus mutans, hence it is evident that shorten the sterilizing time to streptococcus mutans, extend it to deforming chain
The suppression growth of coccus.
6. chlorhexidine acetate nano-emulsion gargle is to Escherichia coli, candida albicans, streptococcus mutans and Bacillus acidi lactici
MIC detection
Experiment material and packet：0.5% chlorhexidine acetate nano-emulsion gargle group (writes a Chinese character in simplified form CNE, preparation using the present invention
Technique, optimized prescription be：Tween 80：Propane diols：IPM (19.2%:4.8%:6%, i.e. specific embodiment 1), acetic acid
Chlorohexidene concentration 0.5% and water 56.5%, peppermint oil 5%).Compound chlorhexidine acetate micro emulsion disinfectant group (is write a Chinese character in simplified form, FCHME, is joined
According to Chinese patent CN103081940A embodiments 1, major pharmaceutical component：Oneself is fixed for 0.5% acetic acid, 0.5% benzalkonium bromide, and 25%
RH40,8.3% propane diols, 2.7% ethyl acetate)；Chlorhexidine acetate micro emulsion disinfectant group (is write a Chinese character in simplified form, CHME, with reference to Chinese patent
CN103081940A embodiments 1, major pharmaceutical component：0.5% acetic acid oneself determine 25%RH40,8.3% propane diols, 2.7% acetic acid
Ethyl ester；Preparation method is according to the formula each component and its use disclosed in the application for a patent for invention of Application No. 96115261.3
Amount, and the chlorhexidine acetate micro emulsion disinfectant prepared according to its disclosed method)
Combine early period preliminary experiment grope, by chlorhexidine acetate nano-emulsion gargle, chlorhexidine acetate micro emulsion disinfectant group,
Chlorhexidine acetate micro emulsion disinfectant group is diluted to containing chlorhexidine acetate 1,2,4,8,16,32,48,64ug/ml respectively.By large intestine bar
Bacterium (ATCC 8099), candida albicans (ATCC 10231), streptococcus mutans (ATCC700610), Bacillus acidi lactici culture
Bacterium solution is adjusted to OD=1.0.Draw and dilute 1 with BHI:10000 times of bacterium solution 1.8ml, by above-mentioned various concentrations chlorhexidine acetate
Solution 0.2ml, is put into 37 DEG C of micro- aerobic incubators, cultivates 24h, 200ul liquid is removed per hole, with microplate reader in 595nm wavelength
Detect its absorption values in place.
Chlorhexidine acetate nano-emulsion gargle is to Escherichia coli, candida albicans, streptococcus mutans, Bacillus acidi lactici
Testing result Fig. 8 of MIC.Fig. 8 results find out that nano-emulsion gargle of the invention is to Escherichia coli, candida albicans, deformation
The MIC of streptococcus and Bacillus acidi lactici is respectively 0.8ug/ml, 3.2ug/ml, 0.4ug/ml, 3.2ug/ml.Chlorhexidine acetate folk prescription
To Escherichia coli, candida albicans, streptococcus mutans and Bacillus acidi lactici it is 3.2ug/ respectively with the micro emulsion disinfectant of compound
Ml and 1.6ug/ml, 6.4ug/ml and 4.8ug/ml, 1.6ug/ml and 0.8ug/ml, 6.4ug/ml and 3.2ug/ml.The present invention
Nano-emulsion gargle be acetic acid respectively to Escherichia coli, candida albicans, streptococcus mutans and Bacillus acidi lactici antibacterial effect
Chlorhexidine folk prescription and 4 times of compound micro emulsion and 2 times, 2 times and 1.5 times, 4 times and 2 times, 2 times and 1 times.
7. suppression production acid of the chlorhexidine acetate nano-emulsion on streptococcus mutans influences
By chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion gargle group be diluted to respectively containing chlorhexidine acetate 2,
4、6、8ug/ml.The ATCC700610 bacterium solutions cultivated are adjusted to OD=1.0.By ATCC700610 bacterium solutions BHI Liquid Cultures
Base is drawn 1.8ml after diluting 1000 times and is put into 24 porocyte culture plates, by above-mentioned various concentrations chlorhexidine acetate solution
0.2ml, is put into 37 DEG C of micro- aerobic incubators, cultivates 24h, 1ml liquid is removed per hole, with precise digital display acidometer pH value determination.
Chlorhexidine acetate nano-emulsion, on the influence of streptococcus mutans acid production as a result, seeing Fig. 9 a.It can be seen that from Fig. 9 a,
Chlorhexidine acetate nano-emulsion and its aqueous solution are 0.2ug/ml, and to streptococcus mutans production acid, there was no significant difference.When concentration increases
4ug/ml is added to, the suppression acid production of chlorhexidine acetate nano-emulsion is obvious, and chlorhexidine acetate aqueous solution is increased to when concentration
8ug/ml could substantially suppress the production acid of streptococcus mutans just, and the nano-emulsion of blank compares, and acid nothing is produced to streptococcus mutans and is appointed
What significantly inhibits effect.
8. chlorhexidine acetate nano-emulsion, which forms slightly solubility exocellular polysaccharide streptococcus mutans, to be influenced
By chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion gargle group be diluted to respectively containing chlorhexidine acetate 2,
4、6、8ug/ml.The ATCC700610 bacterium solutions cultivated are adjusted to OD=1.0.By ATCC700610 bacterium solutions BHI Liquid Cultures
Base is drawn 1.8ml after diluting 1000 times and is put into 24 porocyte culture plates, by above-mentioned various concentrations chlorhexidine acetate solution
0.2ml, is put into 37 DEG C of micro- aerobic incubators, cultivates 48h, collects supernatant, and under the conditions of 6000g, centrifuges 15min, 4 DEG C, abandons
Supernatant.Add 0.5mol/L NaOH 1ml to repeat to mix, under the conditions of 6000g, centrifuge 15min, wash 2 times, precipitation is with 2ml's
After 0.1M NaOH dissolvings its content is detected with Phenol sulfuric acid procedure.Each sample is repeated 3 times.
Chlorhexidine acetate nano-emulsion, the slightly solubility exocellular polysaccharide of streptococcus mutans is influenced as a result, seeing Fig. 9 b.From Fig. 9 b
It can be seen that, chlorhexidine acetate nano-emulsion and its aqueous solution are 0.2ug/ml, and sour slightly solubility exocellular polysaccharide is produced to streptococcus mutans
There was no significant difference.When concentration increases to 4ug/ml, the inhibitory action of chlorhexidine acetate nano-emulsion is obvious, and chlorhexidine acetate water
Solution could substantially suppress the generation of the exocellular polysaccharide of streptococcus mutans just when concentration increases to 8ug/ml, and the nano-emulsion of blank
Control, produces slightly solubility exocellular polysaccharide to streptococcus mutans and significantly inhibits effect without any.
By it is produced acid, slightly solubility exocellular polysaccharide observation confirms that, chlorhexidine acetate nanometer can suppress production acid, born of the same parents
The generation of exo polysaccharides.
9. chlorhexidine acetate nano-emulsion forms biofilm formation to streptococcus mutans to be influenced
(1) streptococcus mutans biofilm formation amount
Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion gargle group are diluted to containing chlorhexidine acetate respectively
500ug/ml.The ATCC700610 bacterium solutions cultivated are adjusted to OD=1.0.By ATCC700610 bacterium solutions BHI fluid nutrient mediums
2ml is drawn after 10000 times of dilution and is put into 24 porocyte culture plates, is put into 80%N2,20%CO2, micro- aerobic training under the conditions of 37 DEG C
48h is supported to take out.Abandoning supernatant, adds 0.2ml and is made with the nano-emulsion gargle and aqueous solution of the chlorhexidine acetate containing 500ug/ml
With 30min, equipped with being cleaned 2~3 times, drying at room temperature 30min in 96 orifice plate of PBS standards, 0.1% crystal violet is added per hole
200 μ l, 15min dyeing, are cleaned 3 times, drying at room temperature 10min with PBS same methods.95% ethanol, 200 μ l/ holes are being added in 96
In orifice plate, 30min is shaken on micro oscillator, in wavelength is its OD numerical value of detection at 570nm with microplate reader, is repeated 3 times per hole,
Compareed using the hole without bacteria suspension as radix.
Chlorhexidine acetate nano-emulsion, influences as a result, seeing Fig.10a the biofilm formation amount of streptococcus mutans.From Figure 10
It can be seen that, substantially reduced with same amount chlorhexidine acetate nano-emulsion and aqueous solution effect streptococcus mutans biofilm biomass, and vinegar
Sour chlorohexidene aqueous solution is substantially higher than the amount between blank control group.And chlorhexidine acetate nano-emulsion, chlorhexidine acetate aqueous solution pair
The biofilm formation amount of blank control group produces notable difference.
(2) the biological leptonomorphology of its suppression of scanning electron microscopic observation
Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion gargle group are diluted to 2ug/ containing chlorhexidine acetate respectively
ml.The ATCC700610 bacterium solutions cultivated are adjusted to OD=1.0.ATCC700610 bacterium solutions BHI fluid nutrient mediums are diluted
Draw 1.8ml after 10000 times to be put into 24 porocyte culture plates, by above-mentioned various concentrations chlorhexidine acetate solution 0.2ml.It is put into
80%N2,20%CO2, micro- aerobic culture 24h and 48h under the conditions of 37 DEG C.Its plastic sheet is carefully taken out, with 2.5% glutaraldehyde
It is fixed, successively with respectively dehydration 1 time of 50%, 70%, 80%, 90% alcohol, then with 100% dehydration of alcohol 2 times, isoamyl acetate
Dehydration 2 times, is 5min every time, and CO2 is freeze-dried 5h, and carrier surface plates bronze, SEM observations under vacuum.
Chlorhexidine acetate nano-emulsion gargle is to the biofilm formation apperance of streptococcus mutans as a result, seeing that Figure 10 b- scheme
10e.It can be seen that from figure, with chlorhexidine acetate nano-emulsion gargle and aqueous solution the effect deformation chain of same amount chlorhexidine acetate
Coccus biofilm formation cosmetic variation finds that nano-emulsion gargle density is well below it after 2000,4000,8000 times of amplifications
Aqueous solution, but nano-emulsion gargle and aqueous solution are to no significant difference between the form of biomembrane.
(3) atomic force microscope observation is to biomembrane three-dimensional structure morphology
Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion gargle group are diluted to 2ug/ containing chlorhexidine acetate respectively
ml.The ATCC700610 bacterium solutions cultivated are adjusted to OD=1.0.ATCC700610 bacterium solutions BHI fluid nutrient mediums are diluted
Draw 1.8ml after 10000 times to be put into 24 porocyte culture plates, by above-mentioned various concentrations chlorhexidine acetate solution 0.2ml.It is put into
80%N2,20%CO2, micro- aerobic culture 48h under the conditions of 37 DEG C.Its plastic sheet is carefully taken out, is gently cleaned with sterile PBS
3 times, naturally dry, with Atomic Mechanics microscope direct observing.All images are handled by automatic smoothing to eliminate the slow side of sweeping
To low frequency noise.Calculate its arithmetic average root, mean square deviation, maximum height, degree of skewness, steepness.
Streptococcus mutans biomembrane is acted on high-resolution atomic force microscope Dichlorodiphenyl Acetate chlorohexidene nano-emulsion and aqueous solution
Observe and three-dimensional reconstruction the result is shown in Figure 11 a- Figure 11 e.From the X-Y scheme of Figure 10, it can be seen that the biology of nano-emulsion gargle
Film thickness is significantly lower than aqueous solution.Figure 11 graphics then finds out that the density for the biomembrane that nano-emulsion gargle is formed is significantly lower than
Its aqueous solution.His arithmetic average root, mean square deviation, maximum height, nano-emulsion gargle are below its aqueous solution, and degree of skewness and
Steepness is then opposite.
The forming amount, appearance and three-dimensional structure of its biomembrane are proved, chlorhexidine acetate nano-emulsion substantially suppresses to deform chain
Coccus formed amount and meanwhile suppress density and the thickness to be formed, but to its biofilm formation streptococcus mutans form without produce it is bright
It is aobvious to change.
(10) chlorhexidine acetate nano-emulsion causes streptococcus mutans the internal pharmacodynamic study of rodents model
Experiment material and packet：Experiment is divided into：Model control group, blank nano-emulsion group, 2mg/ml chlorhexidine acetate nanometers
Newborn gargle group, 5mg/ml chlorhexidine acetate nano-emulsion gargle groups, 2mg/ml chlorhexidine acetate aqueous solution groups, 5mg/ml acetic acid
Chlorohexidene suspension group.Three week old, 60 SPF females rats (Chongqing Institute of Chinese Medicine) are randomly divided into 6 groups, every group 10
Only, the free water under SPF environment is raised, preceding chow diet is infected, 2000 carious tooth feeds of Keys is used after infection.In
State's patent (a kind of streptococcus mutans infection and cariogenic method for building animal model, ZL 201210558790.6) carries out, first continuous
Miscellaneous bacteria is removed with 1% chloramphenicol, 1% ampicillin, 1% carbenicillin within 3 days.After antibiotic is removed, all rats are anesthetized
Afterwards, oral cavity instills 1 × 109CFU/ml, 700610 bacterium solutions of 0.2ml.2 times a day, for three days on end.The 5th day after infection, except model
Outside group animal, remaining every group of animal is anesthetized the different solutions for instilling 0.2ml, 2 times a day, continuous 28 days.Upon administration the 14th
It and 28 days, rat oral cavity is taken after anesthesia with aseptic cotton carrier, it is instilled light salivas with 100 times of dilution 5ul of sterile saline
Culture medium, micro- aerobic condition of culture culture 3 days, is counted with bacterial colony counting instrument.The 70th day after infection, all animals were condemned to death,
Its tooth evaluates the carious tooth of each group animal with Keys (1958) points-scoring system.
14 days and 28 days after successive administration, the result is shown in figure figure .12a for rat oral cavity clump count.Twice daily continuous 14
It and administration in 28 days, the mouth clump count of 2mg/ml and 5mg/ml CNE are substantially less than the CHX of same concentrations.Meanwhile with administration
The extension of time, the clump count in oral cavity are reduced.To the carious tooth appraisal result of all animals, Figure 12 b to Figure 12 d are seen.Can from figure
Find out, the score of the enamel caries of all drug-treated group animals is substantially less than blank nano-emulsion group and model control group.2mg/ml
The fraction of enamel caries, shallow dental caries and middle dental caries with 5mg/ml CNE is the detailed CHX for being less than same concentrations.In addition, as medicine is dense
Degree increase, the enamel caries of its carious tooth, shallow dental caries and middle dental caries fraction are decreased obviously.The representative graph figure of its every group of animal teeth carious tooth can be with
Find out, green arrow represents surface (enamel caries) lesion；Blue arrow represents slight dentine (shallow dental caries) lesion；Red arrow
Indicate moderate dentine (middle dental caries) lesion.It is it can be recognized from fig. 12 that also obvious with the carious tooth degree of 2mg/ml and 5mg/ml CNE
Less than the CHX and control group of same concentrations.Therefore, this is as a result, it was confirmed that CNE can improve antibacterial effect and the reduction of this medicine
Carious tooth degree.
(11) destruction research of the chlorhexidine acetate nano-emulsion to streptococcus mutans cell membrane
Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion gargle group are diluted to 2ug/ containing chlorhexidine acetate respectively
ml.The ATCC700610 bacterium solutions cultivated are adjusted to OD=1.0.ATCC700610 bacterium solutions BHI fluid nutrient mediums are diluted
Draw 1.8ml after 10000 times to be put into 24 porocyte culture plates, by above-mentioned various concentrations chlorhexidine acetate solution 0.2ml.It is put into
80%N2,20%CO2, micro- aerobic culture 48h under the conditions of 37 DEG C.By nutrient solution 6000g, with 2.5% glutaraldehyde after 10min centrifugations
It is fixed.After sample is fixed with 2% (w/v) osmium 0.1M PBS at room temperature 1 it is small when, washed three times with identical buffer solution,
Then it is dehydrated in a series of ethanol solution (that is, 50%, 70%, 80%, 90%, 95% and 100%).Each sample is embedded into
Oere horse ultramicrotome is used in epoxy resin and reduction, with uranyl acetate stain and alkaline lead citrate and use
Tecnai-10TEM is observed.
Streptococcus mutans cell membrane damage, is shown by transmission electron microscope observation, in Figure 13 a to Figure 13 d.Amplifying
3.9 ten thousand times, more obvious loophole carries out processing with observing (Figure 13 a) than CHX (Figure 13 b) to Streptococcus mutans after CNE.
9.3 ten thousand times, CHX shows equally distributed endochylema that is complete, clearly identifying cell membrane.However, as CNE shows to change
With destroy cell film process cell, as depicted in fig. 13 a.The appearance of cytoplasma membrane becomes from cell membrane local detachment.Compare, with
It is serious (Figure 13 a and Figure 13 c) that CHX, which observes the damaged degree,.Cell membrane interruption is more serious in 0.2 μ g/mL CNE
Group and CHX.These results indicate that CNE shows the streptococcus mutans of stronger destruction membrane structure than the CHX's of same concentration
Ability.
ATC700610 bacteriums bacterium is cultivated (1 × 106CFU/ milliliters) culture when 24 well culture plates 4 are small with CNE and
The μ of CHX0.2,0.4 and 0.8 g/ml chlorhexidine acetates contents assess the integrality of cell membrane.By the bacterium 6000rpm of culture
Centrifuge 10min, precipitation add 200ul physiological saline solution, take its 5ul with micro ultraviolet specrophotometer 260nm with
280nm measures its absorption values.
The integrity result of cell membrane is shown in Figure 13 e and Figure 13 f.It can be seen that from Figure 13 e, 0.4 and 0.8 μ g/mL CNE
The 260nm extinction number of degrees, be significantly higher than the CHX (P=000, P=0.001, P ＜ 0.01) of same concentrations.Due to breaking for cell membrane
Bad and damage, 0.4 and 0.8 μ g/mL CNE are from the nucleic acid amount that cell membrane leaks apparently higher than the CHX of same concentrations.Can from Figure 13 f
Find out, the 280nm extinction number of degrees of 0.4 and 0.8 μ g/mL CNE, be significantly higher than CHX (P=000, P=0.001, the P of same concentrations
＜ 0.01).Due to the destruction and damage of cell membrane, 0.4 and 0.8 μ g/mL CNE from the protein content that cell membrane leaks apparently higher than
The CHX of same concentrations.Due to the destruction and damage of cell membrane, the CNE of 0.4 and 0.8 μ g/mL is in 260 and 280nm absorbances
1.75 times, 1.78 times and 6.25 times of its aqueous solution and 4.43 times.The studies above the result shows that, chlorhexidine acetate nano-emulsion is gargled
Liquid is the formation by destroying membrane structure and biomembrane, so as to improve the antibacterial activity to streptococcus mutans.
The present invention passes through the acetic acid using Tween 80/propane diols/IPM as representative to solubility experiment, successfully filtering out
Chlorohexidene nano-emulsion gargle, is confirmed with transmission electron microscope, high speed centrifugation, current potential and Particle Size Analyzer, HPLC, successfully prepares grain
Footpath is in 1-100nm, and envelop rate is more than 90%, and drugloading rate is 0.5% chlorhexidine acetate nano-emulsion gargle.At the same time to its room temperature
Content, granularity, current potential, pH and its appearance research placed 1 year confirm that nano-emulsion gargle quality is extremely stable.Pass through body
Outer MIC, MBC, antibacterial and sterilization dynamic test and the scoring of internal carious tooth confirms that nano-emulsion gargle is to streptococcus mutans
Activity with notable in-vitro antibacterial.Acid, slightly solubility exocellular polysaccharide and biofilm biomass, appearance and three-dimensional structure research are produced to it
It was found that nano-emulsion is gargled, liquid energy significantly inhibits its production acid, slightly solubility exocellular polysaccharide and biofilm formation amount, change for forming biomembrane
Shape hammer strain density, quantity and thickness, destroy bacterial cell membrane.
All bibliography referred in invention are incorporated herein by reference, just as each document quilt
It is individually recited as reference, without being further elaborated on, it is believed that use content disclosed above, people in the art
Member can apply to greatest extent, chlorhexidine acetate pharmaceutical preparation stability of the such equivalent forms in addition to nano-emulsion bibliography,
The application the appended claims can not possibly be reached at the same time in terms of drug delivery amount, envelop rate, preparation process and drug effect to be limited
Fixed scope.In addition, preferred embodiment above should be understood to be merely illustrative of, rather than limit in any way
The scope of the present invention.The above is only the preferred embodiment of the present invention rather than limiting the scope of the invention, should
Point out, for those skilled in the art, without departing from the principle of the present invention, can be to the present invention
Technical solution is modified or equivalent substitution, these modifications or equivalent substitution also should be regarded as protection scope of the present invention.
Claims (2)
1. a kind of antibacterial, the chlorhexidine acetate nano-emulsion gargle for preventing and treating carious tooth, it is characterised in that by following parts by weight
Raw material be made：0.2~1.5 part of chlorhexidine acetate, 10~30 parts of Tween-80,4.8~15 parts of 1,3- propane diols, IPM 3~
34.45~69.0 parts of 20 parts, 0.05~1 part of oral cleansing lotion and distilled water；The chlorhexidine acetate nano-emulsion gargle, is used
Its particle diameter is 1~100nm after water infinite dilution.
2. antibacterial, the chlorhexidine acetate nano-emulsion gargle of prevention and treatment carious tooth, its feature exist according to claim 1
In the oral cleansing lotion is：Any of natural peppermint, natural borneol, peppermint oil, menthol or its combination.
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